摘要
目的探讨低浓度氟化钠对人牙髓细胞(human dental pulp cells,hDPCs)成骨/成牙本质分化的影响。方法本研究已通过单位伦理委员会审查批准。原代培养hDPCs,采用MTT法检测不同浓度氟化钠对hDPCs增殖的影响;选取合适浓度的氟化钠加入成骨/成牙本质分化诱导培养液中,对hDPCs进行体外诱导,通过茜素红染色检测hDPCs成骨/成牙本质分化能力的变化,RT⁃qPCR检测分化相关基因的mRNA表达;同时通过RT⁃qPCR和Western blot检测hDPCs成骨/成牙本质分化过程中内质网应激相关基因的表达。结果低浓度氟化钠(0.1 mmol/L)在体外可刺激hDPCs增殖,高浓度氟化钠(5~10 mmol/L)可抑制hDPCs增殖(P<0.05)。选取0.1 mmol/L氟化钠体外混合成骨/成牙本质分化诱导培养后hDPCs的茜素红染色增加,成骨/成牙本质分化相关基因牙本质涎磷蛋白(dentin sialophosphoprotein,DSPP)、骨涎蛋白(bone sialoprotein,BSP)和骨钙蛋白(osteocalcin,OCN)mRNA表达水平升高(P<0.05)。同时在此过程中RT⁃qPCR检测出mRNA水平hDPCs内质网应激相关基因:剪切x盒结合蛋白1(splicing x⁃box binding protein⁃1,sXBP1)、葡萄糖调节蛋白78(glucose⁃regulated protein 78,GRP78)以及活化转录因子4(activating transcription factor 4,ATF4)表达升高(P<0.05);Western blot检测出氟化钠混合成骨/成牙本质分化培养后细胞磷酸化真核起始因子⁃2α(phosphorylated eukary⁃otic initiation factor⁃2α,p⁃eIF2α)、磷酸化蛋白激酶样内质网激酶(phosphorylated the RNA⁃activated protein kinase⁃like ER⁃resident kinase,p⁃PERK)和ATF4蛋白表达增加(P<0.05)。结论低剂量氟化钠促进人牙髓细胞的成骨/成牙本质分化并伴有内质网应激水平的升高。
Objective To study the effect of low concentrations of sodium fluoride on the osteogenic/odontogenic differentiation of human dental pulp cells(hDPCs)in vitro.Methods This study was reviewed and approved by the Ethics Committee.hDPCs were cultured using a modified tissue explant technique in vitro.The effects of different con-centrations of sodium fluoride on the proliferation of hDPCs were measured by methylthiazol tetrazolium(MTT)assay.Appropriate concentrations were added to the osteogenic/odontogenic differentiation induction medium,and the cells were induced in vitro.Alizarin red S staining was used to detect the osteoblastic/odontogenic differentiation ability of the cells,and the mRNA expression of the key differentiation factors was detected by RT-qPCR.Moreover,the expres-sion of key molecules of endoplasmic reticulum stress(ERS)was detected by RT-qPCR and Western blot.The data were analyzed with the SPSS 18.0 software package.Results Low concentration of NaF(0.1 mmol/L)could stimulate cell proliferation in vitro,while a high concentration(5-10 mmol/L)could inhibit cell proliferation(P<0.05).According to the literature and the experimental data,0.1 mmol/L NaF was selected as the following experimental concentration.The levels of alizarin red S staining were increased after NaF induction of mixed osteogenic/odontogenic differentiation in vi-tro.The mRNA expression levels of key molecules for osteogenic/odontogenic differentiation,dentin sialophosphoprotein(DSPP),bone sialoprotein(BSP)and osteocalcin(OCN),were increased(P<0.05).The mRNA levels of ERS markers(splicing x-box binding protein-1(sXBP1),glucose-regulated protein 78(GRP78)and activating transcription Factor 4(ATF4)were increased in NaF-treated cells.The protein expression levels of key ER stress molecules(phosphorylated RNA-activated protein kinase-like ER-resident kinase(p-PERK),phosphorylated eukaryotic initiation factor-2α(p-eIF2α)and ATF4)were higher in NaF-treated cells.Conclusion A low concentration of NaF promotes the osteogenic/odontogenic differentiation of hDPCs and increases the level of ER stress.
作者
李莉芬
韩俊力
江龙
LI Lifen;HAN Junli;JIANG Long(Department of General Dentistry,Shanghai Ninth People's Hospital,Col-lege of Stomatology,Shanghai Jiao Tong University School of Medicine&National Center for Stomatology&National Clinical Research Center for Oral Disease&Shanghai Key Laboratory of Stomatology&Shanghai Research Insititute of Stomatology,Shanghai 200011,China)
出处
《口腔疾病防治》
2024年第1期22-28,共7页
Journal of Prevention and Treatment for Stomatological Diseases
基金
上海市青年科技英才扬帆计划(21YF1424100)
上海交通大学医学院附属第九人民医院基础研究助推计划(JYZZ183)。
关键词
人牙髓细胞
氟化钠
增殖
成骨/成牙本质分化
内质网应激
剪切X盒结合蛋白1
活化转录因子4
葡萄糖调节蛋白78
蛋白激酶样内质网激酶
真核起始因子⁃2α
human dental pulp cell
sodium fluoride
proliferation
osteogenic/odontogenic differentiation
endoplasmic reticulum stress
splicing xbox binding protein 1
activating transcription factor 4
glucose⁃regulated protein 78
RNA⁃activated protein kinase⁃like ER⁃resident kinase
eukaryotic initiation factor⁃2α
